Abstract

Several recent studies have employed warm-water treatment of diatom cells to extract nominally bound extracellular polymeric substances. Where examined, the dominant neutral sugar in these extracts was glucose. In the present study, we sought to characterize the structure of the glucose-rich polymers in the water extracts of diatoms and to determine the origin of these polymers. The marine diatoms surveyed were Phaeodactylum tricornutum, Cylindrotheca fusiformis, Craspedostauros australis and Thalassiosira pseudonana. A freshwater species, Pinnularia viridis, was also investigated for the dye labelling experiments. Freshly harvested marine diatoms were extracted with water at 308C for 1 h. Constituent monosaccharide analyses showed that glucose was the dominant neutral sugar (80 - 95 mol% of the total) in the extracts from three marine species, whereas the P. tricornutum extract contained predominantly ribose, galactose and glucose, and was inferred to be enriched in low-molecular-weight components. Linkage analysis of the constituent monosaccharides and proton nuclear magnetic resonance spectroscopy showed that the glucose in these extracts was derived primarily from 1,3-b-D-glucan. Immunocytochemistry with a monoclonal anti-1,3-b-D-glucan antibody confirmed that the glucan was localized in the vacuoles of diatom cells preserved by freeze-substitution. Nearly all diatom cells incubated with a fluorescent dye, DiBAC4(3), during warm water treatment at 308C or 608C incorporated the dye, demonstrating that the membrane integrity of the diatoms was compromised and supporting the contention that intracellular glucan was released during the treatment. In light of these data, the extracellular glucans of diatoms reported in some previous studies are re-interpreted as intracellular chrysolaminaran.